Cytochrome c oxidase (COX) deficiency is the most frequent cause of mitochondrial neuromyopathies in humans. CQXdeficiency is associated with heterogeneous clinical phenotypes, including Leigh syndrome, muscle weakness and encephalomyopathy. A better understanding of COX biogenesis is essential for elucidating the molecular basis underlying this group of diseases. The main objective of the proposed project is to investigate COX assembly using the yeast Saccharomyces cerevisiae as a model. The yeast paradigm will be instrumental in clarifying the molecular basis of human COX deficiencies. Eukaryotic COX is formed of 12-13 different polypeptides. The three subunits forming the catalytic core are encoded by mitochondrial DNA. The other subunits are all products of nuclear genes that are translated oncytoplasmic ribosomes and imported into mitochondria. Biogenesis of the functional complex requires the additional function of more than 20 COX-specific assembly genes. We have obtained data revealing the existence of a regulatory mechanism by which the synthesis of Cox1 p, a mitochondrially encoded catalytic subunit of COX, is regulated by the availability of its assembly partners. Mss51p is a nuclear encoded COX1 mRNA specific translational activator which plays a key role in this regulation. MSS51 acts as an extragenic suppressor of shyl mutants, our yeast model of Leigh syndrome. Mss51p interacts with the 5'untranslated region of COX1 mRNA, activating synthesis of Coxlp. MSS51p also binds newly synthesized Coxlp, suggesting other roles in Coxlp biogenesis (elongation, membrane insertion or maturation by addition of prosthetic groups). The goal of the proposed studies is to identify and characterize multiple and specialized domains in MSS51p involved in different functions. Several specific aims will be pursued. 1)We will further characterize the selected mss51 point mutants obtained by chemical random mutagenesis in terms of Coxlp expression, COX assembly and function. 2) We have identified the presence of two CPX domains located in the N1 terminus of Mss51p. These domains are present in heme lyases and could be used by MSS51p to bind heme a3 and insert it into Coxlp. Site directed mutagenesis will be used to characterize these domains for their relevance in Cox1 p biogenesis, COX assembly and function. 3) Mss51 p is part of a high molecular weight complex. To complete the picture of how MSS51p regulates Cox1 p biogenesis, the partners of Mss51p in this complex will be identified. In summary, the yeast paradigm will be exploited by biochemical and genetic means to gain a complete understanding of the function of Mss51p, how it contributes to regulate Coxlp biogenesis, and to help clarifying the molecular basis of human COX deficiencies.